Aryl Hydrocarbon Receptor Activation Suppresses EBF1 and PAX5 and Impairs Human B Lymphopoiesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.

TCDD: an environmental immunotoxicant reveals a novel pathway of immunoregulation--a 30-year odyssey.

I was honored to be the keynote speaker at the 30th Annual Society of Toxicologic Pathology Symposium "Toxicologic Pathology and the Immune System." I had the opportunity to reminisce about events in the 1970s that set the stage for the birth and subsequent growth of the field of immunotoxicology and to summarize my research career that has spanned the past 40 years as well. An initial focus on the immunotoxicity of pentachlorophenol led my laboratory into the aryl hydrocarbon receptor (AHR) field and the study of its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). My research career has been devoted to trying to elucidate the immunological basis of TCDD's profound immunosuppressive activity that is mediated by activation of AHR. In recent years, my laboratory has focused on the role of CD4(+ )T cells as targets of TCDD, and we were the first to describe the induction of AHR-dependent regulatory T cells (Tregs). The ability to induce Tregs using an exogenous AHR ligand to activate the AHR-Treg pathway represents a novel approach to the prevention and/or treatment of autoimmune disease. We are currently searching for such ligands.

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs stable establishment of oral tolerance in mice.

The toxic environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunomodulatory chemical. TCDD activates the aryl hydrocarbon receptor (AhR) and suppresses peripheral humoral and cellular adaptive immune responses. Though the major route of uptake is via food, little is known until now on the immunotoxic effects of TCDD on the gut-associated lymphoid tissue. We show here that AhR is strongly expressed along the small intestine, especially in intestinal epithelial cells (IEC). The AhR marker gene cyp1a1 is induced in IEC by oral TCDD exposure. We asked how TCDD affects oral tolerance, a unique function of mucosal immunity. C57BL/6 mice were injected with 10 µg/kg body weight TCDD and fed with ovalbumin (OVA) in a high-dose tolerization protocol. Mice were immunized and boosted with OVA on days 12, 23, and 55 after tolerization. Five of 14, 6 of 15, and 13 of 14 TCDD-treated mice generated OVA-specific immunoglobulin (Ig)G1 antibodies after the first, second, and third immunization with OVA, respectively. Only one mouse harbored anti-OVA IgG1 antibodies in the control group even after the third immunization with OVA. OVA-specific IgA in fecal samples of tolerized and TCDD-exposed mice could be detected at the levels of nontolerized mice, whereas completely absent in tolerant control mice. Correlated to this, we found in TCDD-treated mice an increase in interleukin-6 producing CD103+ dendritic cells (DC) present in the gut-draining mesenteric lymph nodes (MLN) and a small increase in the frequency of Th17 cells. Neither the frequencies nor the absolute numbers of immune cells in the lamina propria (LP) or in intraepithelial lymphocytes were changed by TCDD treatment. Our data not only have implications for food allergies in settings of environmental exposure but also raise concerns regarding the harmlessness of overdosing potential AhR agonist in food, which needs to be studied further.

Children's immunology, what can we learn from animal studies (3): Impaired mucosal immunity in the gut by 2,3,7,8-tetraclorodibenzo-p-dioxin (TCDD): a possible role for allergic sensitization.

We have recently reported breakdown of mucosal immunity in the gut by tetraclorodibenzo-p-dioxin (TCDD). That is, single oral administration of low dose 2,3,7,8-TCDD resulted in a marked decrease in IgA secretion in AhR-dependent manner and impaired oral tolerance in the gut. In the present study, we found TCDD exposure by breast feeding also resulted in decreased level of IgA in the gut. Ig production by B cells by LPS stimulation was not affected by TCDD administration. Instead, particular chemokine receptor expression on B1 cells, a major cell source for intestinal IgA antibody, was decreased in mice treated with TCDD. In consistence with this observation, B1, but not B2 cells from TCDD treated mice showed impaired chemotaxis towards B lymphocyte chemokine (BLC)/CXCL13. In contrast, chemotaxis of intestinal dendritic cells (DCs) towards secondary lymphoid-tissue chemokine (SLC)/CXCL19 was not impaired in mice treated with TCDD. Furthermore, there was no change in the number and profile of intestinal microflora in TCDD-treated mice. These results indicate that TCDD exposure by breast feeding results in breakdown of intestinal mucosal immunity of pups and that it may be attributed in part to impaired B1 cell migration from the peritoneal cavity to intestinal mucosa.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation of alloreactive CD8+ T cells toward a regulatory T cell phenotype by a mechanism that is dependent on aryl hydrocarbon receptor in CD4+ T cells.

Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetracholordibenzo- p-dioxin (TCDD) during an acute graft-versus-host response induces a population of alloreactive donor CD4+CD25+ regulatory T (Treg)-like cells that have potent suppressive activity in vitro. In the present studies, we show that TCDD induced a similar population of donor CD8+CD25+ T-cells with suppressive activity in vitro. Like the CD4+ Treg cells, donor CD8+CD25+ T-cells also expressed higher levels of CD28, glucocorticoid-induced TNFR (GITR) and CTLA-4 along with low levels of CD62L. These TCDD-induced phenotypic changes were not observed if donor T-cells were obtained from AhR-KO mice. When CD4+ and CD8+ donor T-cells from AhR-WT and AhR-KO mice were injected in various combinations into F1 mice, the enhanced expression of CD25 on CD8+ T-cells required AhR in donor CD4+ T-cells, while down-regulation of CD62L required AhR in the donor CD8+ T-cells themselves. Changes in GITR and CTLA-4 on donor CD8+ T-cells were partially mediated by AhR in both T-cells subsets. In contrast, all phenotypic changes in donor CD4+ T-cells were dependent on the presence of AhR in the CD4+ T-cells themselves. These findings suggest that the direct effects of AhR-mediated signaling in CD8+ T-cells are more limited than the direct effects in CD4+ T-cells, and that AhR signaling in CD4+ T-cells may be a unique pathway for the induction of both CD4+ and CD8+ adaptive Treg.

Dioxin immunosensor using anti-2,3,7,8-TCDD antibody which was produced with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate as a hapten.

To detect dioxin using a quartz crystal microbalance (QCM) immunosensor, anti-2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) monoclonal antibodies (MAbs) were produced as types of IgG1 and IgM, with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate (as a hapten) conjugated with bovine serum albumin (dioxin-BSA). Furthermore, ScFv was generated from hybridoma-producing IgG1 MAb. Among these antibodies, ScFv showed excellent capability for dioxin detection using QCM immunosensors.

Quartz crystal microbalance immunosensor for highly sensitive 2,3,7,8-tetrachlorodibenzo-p-dioxin detection in fly ash from municipal solid waste incinerators.

A quartz crystal microbalance (QCM) immunosensor was developed for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD) in environmental pollutants. An anti-TCDD antibody was immobilized on the gold surface of the QCM via chemical coupling, and its immunologic activity was then maintained by treatment with an artificial stabilizing reagent such as poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). A competitive immunoreaction with TCDD conjugated ovalbumin (TCDD-ovalbumin) was used to detect TCDD. A calibration curve was obtained through the competitive immunoreaction, and linearity was shown from 100 ng mL(-1) to 0.1 ng mL(-1). Also, the cross-reactivities of the anti-TCDD monoclonal antibody were thoroughly evaluated with several TCDD derivatives. The relationships between GC-MS, ELISA, and QCM were compared using fly ash samples from a municipal solid waste, which were prepared using an accelerated solvent extractor. For 23 samples, the experimental relationship between the TCDD concentration by QCM vs. the TCDD concentration by ELISA was y= 1.07x + 2.70, r= 0.99, and the TCDD concentration by QCM vs. the toxic equivalent quantity (TEQ) value by GC-MS was y= 2.46x - 14.98, r= 0.89.

Constitutively active aryl hydrocarbon receptor expressed specifically in T-lineage cells causes thymus involution and suppresses the immunization-induced increase in splenocytes.

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the basic helix-loop-helix-PER-ARNT-SIM superfamily. Xenobiotics, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, bind the receptor and trigger diverse biological reactions. Thymocyte development and T cell-dependent immune reactions are sensitive targets of AhR-dependent 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. However, the exact role of the AhR in T cells in animals exposed to exogenous ligands has not been clarified because indirect effects of activated AhR in other cell types cannot be excluded. In this study, we generated transgenic (Tg) mice expressing a constitutively active mutant of AhR under the regulation of a T cell-specific CD2 promoter to examine AhR function in T cells. The mRNAs of the constitutively active mutant of AhR and an AhR-induced gene, CYP1A1, were expressed in the thymus and spleen of the Tg mice. The transgene expression was clearly detected in the thymocytes, CD4, and CD8 T cells, but not in the B cells or thymus stromal cells. These Tg mice had a decreased number of thymocytes and an increased percentage of CD8 single-positive thymocytes, but their splenocytes were much less affected. By contrast, the increase in number of T cells and B cells taking place in the spleen after immunization was significantly suppressed in the Tg mice. These results clearly show that AhR activation in the T-lineage cells is directly involved in thymocyte loss and skewed differentiation. They also indicate that AhR activation in T cells and not in B cells suppresses the immunization-induced increase in both T cells and B cells.

Immunologic effects of dioxin: new results from Seveso and comparison with other studies.

Animal studies indicate that the immune system is one of the most sensitive targets of the toxic effects of 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD). TCDD inhibits immunoglobulin secretion and decreases resistance to bacterial, viral, and parasitic infections in exposed animals. Nearly 20 years after the Seveso, Italy, accident, we measured immunoglobulin and complement plasma levels in a random sample of the population in the most highly exposed zones (n = 62) and in the surrounding noncontaminated area (n = 58). Plasma IgG levels decreased with increasing TCDD plasma concentration (r = -0.35, p = 0.0002). Median IgG concentration decreased from 1,526 mg/dL in the group with the lowest (< 3.5 ppt) TCDD levels to 1,163 mg/dL in the group with the highest (20.1-89.9 ppt) TCDD levels (p = 0.002). The association was significant (p = 0.0004) after adjusting for age, sex, smoking, and consumption of domestic livestock and poultry in multiple regression analysis and persisted after exclusion of subjects with inflammatory diseases and those using antibiotics or nonsteroidal anti-inflammatory drugs. IgM, IgA, C3, and C4 plasma concentrations did not exhibit any consistent association with TCDD levels. We performed a systematic review of all the articles published between 1966 and 2001 on human subjects exposed to TCDD reporting information on circulating levels of immunoglobulins and/or complement components. The literature indicates that the evidence for effects of TCDD on humoral immunity is sparse. Methodologic issues, results, and possible sources of variation between studies are discussed. The possible long-term immunologic effects of TCDD exhibited by the participants of the present study, coupled with the increased incidence of lymphatic tumors in the area of the accident, warrant further investigation.

Recent advances in understanding the mechanisms of TCDD immunotoxicity.

TCDD is a highly immunosuppressive chemical that induces potent suppression of immune responses in laboratory animals. However, apart from the requisite role of the AhR and the identification of bone-marrow-derived cells as critical AhR-expressing targets, the specific cells and the underlying biochemical mechanisms by which TCDD disrupts immunological functions remain unclear. Recent data suggest that a new paradigm for the mechanism of immunotoxic action of TCDD may be more accurate, moving from one focused on the suppression of immune functions to one focused on the inappropriate activation of cells, leading to anergy or death, and the consequent premature termination of the immune response. Enhanced activation of B cells, DC and CD4+ T cells by TCDD has been described as well as the earlier disappearance of the latter two populations from peripheral lymphoid organs. Although much remains to be learned about how inappropriate cellular activation via the AhR induces immune suppression, deducing this mechanism of action and the signaling pathways involved, should lead to new insight into basic mechanisms of immune regulation.

Dioxin detection based on immunoassay using a polyclonal antibody against octa-chlorinated dibenzo-p-dioxin (OCDD).

An enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody against octa-chlorinated dibenzo-p-dioxin (OCDD) is presented. This method is based on a competitive reaction between OCDD and OCDD-HRP (horseradish peroxidase) conjugate against the antibody, whereby OCDD-HRP is detected colorimetrically at 450 nm. The detection limit of OCDD was 0.78 pg mL(-1). Optimizing the reaction conditions of the assay, cross reactivities of some dioxins against the antibody are discussed.

Immunochemical analysis for dioxins--progress and prospects.

Recent developments in antibody design and sample preparation have considerably enhanced the use of enzyme immunoassay (EIA) as an alternative to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of the trace organic pollutants polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in source and environmental samples. The EIA-specific sample preparation technique for solid matrices using a compatible extracting solvent coupled with an assay using a more sensitive antibody permits screening of source and environmental samples to be undertaken with minimal sample preparation. EIA has been validated on an increasing range of environmental and source samples as a cost effective I-TEQ screening tool, exploiting the commonly recognised general strengths of immunoassays such as speed, simplicity, low cost and parallel processing of many samples, greatly reducing the number of samples requiring conventional GCMS analysis. Further research and developmental work is required in the areas of sample extraction and extract cleanup to ensure compatibility with the immunoassay from the outset, and to standardise as far as is possible an assay format relevant to the application of interest, covering sample preparation, sample cleanup, quality control, assay procedure and data analysis.

Serum dioxin and immunologic response in veterans of Operation Ranch Hand.

The authors studied immune response and exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) among veterans of Operation Ranch Hand, the US Air Force unit responsible for the aerial spraying of herbicides in Vietnam from 1962 to 1971. A comparison group of Air Force veterans who served in Southeast Asia but were not involved in spraying herbicides was included. The authors studied delayed-type hypersensitivity skin test responses to Candida albicans, mumps, Trichophyton, and a bacterial antigen made from lysed Staphylococcus aureus. Lymphocyte measurements included total lymphocyte counts; T-cell (CD3, CD4, CD5, and CD8), B-cell (CD20), and NK-cell (CD16 and CD56) subsets; and expression of the activation antigen CD25 on CD3 T cells. The authors quantitated the serum concentrations of immunoglobulin (Ig)A, IgG, and IgM; examined sera for the presence of monoclonal immunoglobulins (M proteins); and looked for a broad range of autoantibodies (rheumatoid factor, antinuclear antibody, smooth muscle autoantibody, mitochondrial autoantibody, parietal cell autoantibody, and thyroid microsomal autoantibodies). They measured the level of dioxin in 1987 or 1992, extrapolated the result to the time of service in Vietnam, and assigned each veteran to one of four exposure categories: Comparison and three Ranch Hand groups (Background, Low, or High). Overall, the authors found no evidence of a consistent relation between dioxin exposure category and immune system alteration.

Immunologic findings in workers formerly exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and its congeners.

One hundred ninety-two workers in a German pesticide factory who were exposed to polychlorinated dibenzodioxins and -furans (PCDD/PCDF) were investigated for former and present diseases and laboratory changes of the immune system. Moreover, in a subgroup of 29 highly exposed and 28 control persons, proliferation studies were performed. In addition to assays such as blood count, immunoglobulins, serum electrophoresis, monoclonal bands, surface markers, autoantibodies, and lymphocyte proliferation, two new methods, the rise of tetanus antibody concentration after vaccination and the in vitro resistance of lymphocytes to chromate, were used to diagnose the morphologic and functional state of the immune system. There was no stringent correlation of actual PCDD/PCDF concentrations with the occurrence of infections or with one of the immune parameters. In addition, outcomes of the tetanus vaccination and the chromate resistance test were not correlated with PCDD/PCDF. However, the chromate resistance of lymphocytes stimulated by phytohemagglutinin of highly exposed persons was significantly lower than that for the control group. These findings indicate that the function of lymphocytes can be stressed and possibly impaired by high exposure to PCDD/PCDF.

Immune cell functions in industrial workers after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: dissociation of antigen-specific T-cell responses in cultures of diluted whole blood and of isolated peripheral blood mononuclear cells.

A comparative analysis was performed of the phenotype and function of peripheral blood leukocytes of two age-matched cohorts of industrial workers in chemical plants, one of which was exposed occupationally to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Median actual TCDD burdens were 116 ng/kg and 4 ng/kg, respectively. The phenotype analysis of peripheral blood mononuclear cells (PBMC) revealed no significant differences in the proportions of CD3, CD4, or CD8+ T lymphocytes, of CD16+ natural killer cells, and of CD19+ B lymphocytes. However, in PBMC of the TCDD-exposed workers; the proportion of CD8+ memory T cells (CD45R0+) was significantly higher, and that of lymphocytes with naive phenotype (CD45RA+) was significantly lower than in PBMC of the control group. Polyclonal and antigen-specific T-cell activation was assessed in parallel in isolated PBMC as well as in diluted whole blood cultures. In both culture systems the polyclonally stimulated cytokine release did not differ significantly between the two cohorts; however, we found a significantly reduced interferon gamma release in diluted whole blood cultures but not in isolated PBMC cultures of the TCDD-exposed cohort when we performed an antigen-specific T-cell stimulation with tetanus-toxoid. Therefore, we propose that exposure of individuals to high doses of TCDD can partially impair in the "blood milieu" those T-cell/monocyte interactions that are essential for antigen-specific T-cell responses, whereas isolated PBMC of the same donors appear functionally less affected.

Development of a highly sensitive enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of polychlorinated dibenzo-p-dioxins.

The development of an enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies for the polychlorinated dibenzo-p-dioxins is described. We previously reported the synthesis of haptens and generation of antibodies for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Antisera were screened with seven different coating antigens (hapten-protein conjugates), including trans-3-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methylpropeno ic acid (VII) and 5-(3,7,8-trichlorodibenzo-p-dioxin-2-yl)penta-trans,trans-2,4-dien oic acid (X). All inhibition screening and optimization studies were conducted using a less toxic surrogate standard for TCDD [2,3,7-trichloro-8-methyl-dibenzo-p-dioxin (TMDD; XVII)] which responded similarly to 2,3,7,8-TCDD in the ELISA. The most sensitive assay from the screening studies [coating antigen VII-BSA, 0.1 microgram/mL, and antiserum 7598 (anti-X-LPH), 1:10,000] was further optimized and characterized. It exhibited an IC50 value of 12 pg/well (240 pg/mL), with working range from 2 to 240 pg/well (40 to 4800 pg/mL). The influence of various physical and chemical factors (time, solvent, detergent) was investigated. The optimized assay was then used to assess cross-reactivity by congeners of halogenated dioxins and related structures. DMSO up to concentrations of 37.5% decreased the IC50 value in the assay, whereas methanol to concentrations of 30% did not lead to improved IC50 values.

Immunomodulatory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin tested by the popliteal lymph node assay.

Drugs and other chemicals that have the potential to induce or exacerbate systemic autoimmune diseases in humans are of great concern. The aim of this study was to examine the immune-disregulating potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by using the popliteal lymph node (PLN) assay. Chlorpromazine (CPZ) was used as a reference compound for 2 reasons: (a) CPZ is known to elicit a positive response in this assay, and (b) CPZ is a structural analogue of TCDD. Male Sprague-Dawley rats were injected subcutaneously with either TCDD or CPZ into the right hind footpad, whereas vehicle alone was injected into the contralateral footpad. Control rats were injected with vehicle in both hind footpads. Animals were sacrificed on day 7, and their PLNs were removed, weighed, and immersed in 10% formalin. The PLN weight index (the weight ratio of right PLN over left PLN) was significantly higher in both CPZ- and TCDD-treated rats than in controls. Histological examinations of PLNs in the CPZ- and TCDD-treated rats revealed similar morphological changes in both groups (e.g., mild follicular hyperplasia with no evidence of an acute inflammatory response). These results indicate that TCDD has the potential to induce or exacerbate autoimmune-like reactions. Results also suggest that drugs may be useful surrogates to study the mechanism of toxicity of environmental chemicals that cannot be administered to humans.

Alternative models for low dose-response analysis of biochemical and immunological endpoints for tetrachlorodibenzo-p-dioxin.

As part of the current reassessment of dioxin, the empirical relationships between administered tetrachlorodibenzo-p-dioxin and selected immune and biochemical endpoints were investigated. A dose-response analysis from the published literature was performed using Linear, Sigmoid-Emax and Power Law functions. The results of this analysis indicate that the use of a wide dose range may bias the interpretation of low-dose phenomenon. The Power Law function was applied exclusively to low-dose subsets enabling estimation of dose response in the low-dose range. Subsequently, high-dose data were fit using Power Law subset analysis. This approach resulted in a change in slope value from low- to high-dose subsets for thymic atrophy, immune suppression, benzo(a)pyrene hydroxylase activity, and ethoxyresorufin-o-deethylase activity. This change suggests that there is a high probability that there is a tissue concentration along the dose-response continuum which results in biological activity from low to high dose. This analysis also demonstrates that the Power Law functional fit to the low-dose data differs quantitatively from the fit to the high-dose data for several noncancer endpoints. Therefore, by using the Power Law function a more accurate and biologically relevant assessment of risk can be produced for non-cancer endpoints.

Phenotypic analysis of spleen, thymus, and peripheral blood cells in aged C57B1/6 mice following long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

A mouse model was used to identify potential biomarkers of exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Female C57B1/6 mice were treated weekly with 0.2 microgram TCDD/kg body weight or vehicle for 14-15 months. Phenotypic analysis by flow cytometry identified the major cell subpopulations in the spleen, thymus, and peripheral blood as defined by the expression of CD4, CD8, B220, and Mac-1 molecules. These subpopulations were further characterized for the expression of I-A, Pgp-1, CD45RB, and/or T cell receptor antigens (CD3, alpha beta, gamma delta). A group of young (4 months old) mice was evaluated concurrently to document immunophenotype alterations associated with aging. Results showed several age-related changes in phenotype distribution in the spleen and blood, but not in the thymus, despite significant age-dependent thymic involution. The age-dependent changes in splenic phenotypes included a decreased frequency of CD4+ cells and a major shift in the frequency distribution from naive T cells to effector and memory T cells as defined by Pgp-1 and CD45RB expression. These phenotypic changes in the spleen due to aging correlated with similar changes in the blood, providing preliminary support for the use of spleen cells as surrogates for blood in the development of biomarkers of immunotoxicity. In comparison to the effects of aging, TCDD treatment produced relatively subtle changes in immunophenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)